Fibrin nanoparticles coupled with keratinocyte growth factor enhance

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Biological and Medical Applications of Materials and Interfaces

Fibrin nanoparticles coupled with keratinocyte growth factor enhance dermal wound healing rate Ismaeel Muhamed, Erin Sproul, Frances S Ligler, and Ashley C. Brown ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21056 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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ACS Applied Materials & Interfaces

Fibrin nanoparticles coupled with keratinocyte growth factor enhance dermal wound healing rate Ismaeel Muhamed 1,2, Erin P. Sproul 1,2, Frances S. Ligler 1,2 and Ashley C. Brown* 1,2 Joint Department of Biomedical Engineering North Carolina State University and the University of North Carolina at Chapel Hill Biomedical Partnership Centre, Building Suite 20081 1060 William Moore Drive Raleigh, NC 27607 Phone: (919) 513-8231 [email protected], [email protected], [email protected] and *corresponding

1

author [email protected]

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, NC 27695 2 Comparative

Medicine Institute, North Carolina State University, Raleigh, NC 27695

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Keywords: Fibrin nanoparticles, tissue regeneration, cell migration, microfluidics, wound healing, biomaterials, cell migration, in vitro 3D wound model and in vivo dermal wound model

Abstract: Expediting the wound healing process is critical for patients chronically ill from nonhealing wounds and diseases such as hemophilia or diabetes or who have suffered trauma including easily infected open wounds. FDA-approved external tissue sealants include the topical application of fibrin gels, which can be 500 times denser than natural fibrin clots. With lower clot porosity and higher polymerization rates than physiologically formed fibrin clots, the commercial gels quickly stop blood loss, but impede the later clot degradation kinetics and thus retard tissue-healing rates. The fibrin nanoparticles (FBN) described here are constructed from physiologically relevant fibrin concentrations that support new tissue and dermal wound scaffold formation when coupled with growth factors. The FBN, synthesized in a microfluidic droplet generator, support cell adhesion and traction generation, and when coupled to keratinocyte growth factor (KGF), support cell migration and in vivo wound healing. The FBN-KGF particles enhance cell migration in vitro greater than FBN alone or free KGF and also improve healing outcomes in a murine full thickness injury model compared to saline, bulk fibrin sealant, free KGF, or bulk fibrin mixed with KGF treatments. Furthermore, FBN can be potentially administered with other tissue-healing factors and inflammatory mediators to improve woundhealing outcomes.


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Introduction Over 6 million Americans suffer from chronic non-healing wounds, and the consequent cost of annual treatment of wounds is $20 billion USD.1,2 The parameters that affect wound healing vary based on the type of injury (laceration, incision, puncture, abrasion, contusion and burns), local and systemic causative factors (dehiscence, infection, foreign body, oxygenation, blood supply, age, gender, hormones, stress, ischemia, diseases, obesity, medication, alcoholism, smoking, nutrition and immunocompromised conditions) and location of wound (associated with movement and function).3 The primary goal of every wound management therapy is to assist wound closure and restore tissue function.4,5 A host of biomaterials have been developed for wound management spanning from synthetic polymer films to naturally derived hydrogels.6 Examples of naturally-derived hydrogels utilized for wound healing applications include fibrin, collagen, and chitosan-based materials. Materials based on fibrin and collagen are particularly attractive because these proteins serve as native scaffolding materials during the normal healing response.7 In this article, we describe the design of a novel colloidal, pre-polymerized fibrin material that can interface with the native fibrin network and support cell migration required for progression through the various stages of wound healing. The physiological wound healing response is a multi-stage phenomenon and begins by preventing excess blood loss with the formation of blood clots composed primarily of polymerized fibrin (hemostasis), followed by leucocyte infiltration (inflammation), tissue reepithelialization (proliferation) with extracellular matrix (ECM) formation, and wound remodelling.2,8–10 The tight regulation and execution of each stage of wound-healing controls the rate of wound repair for effective wound management. The first step of healing post injury is the formation of a fibrin clot that prevents further blood loss. A fibrin clot is an assembly of cross-



linked fibrin protofibrils, which is formed from fibrin monomers (thrombin-cleaved fibrinogen). The linear polymerization of fibrin monomers forms protofibrils, and their lateral assembly forms fibrin fibers. Fibrin is a biodegradable natural component of ECM, provides a scaffold for wound healing, supports cell adhesion, and modulates migration of cells such as leukocytes and fibroblasts, into the wound bed.1,11–15 The linear fibrin polymer has active ligands for integrin receptor adhesion (αIIbβ316,17, αvβ3, αmβ2, α5β1 , αvβ5 , αIIbβ318,19) and thereby assists cell spreading and re-epithelialization. Since the local synthesis of fibrin at a wound zone is critical for healing, fibrin gels have been used externally as hemostatic agents, tissue sealants, and wound dressings.1,20–23 Fibrin-based materials are attractive because they are biocompatible, biodegradable, and useful for drug delivery, growth factor activation, cell adhesion, and scaffold development.1,13,14 A cascade of enzymatic events modulates the formation of fibrin, which culminates in the activation of fibrinogen by thrombin. The relative concentrations of fibrinogen and thrombin modulate the polymerization rate of the clot and the resulting clot structure, including porosity, fiber thickness, and fiber alignment. Additionally, fibrinolysis (clot degradation) is influenced by clot structure,4,24 and the availability of thrombin, additional coagulation cofactors and plasmin inhibitors. The dominant enzyme that degrades fibrin is plasmin. Even though the generation and degradation of fibrin involves multiple biomolecular checkpoints, the balance between fibrin polymerization and degradation at wound zones is important to achieve physiological healing kinetics. Because fibrin and thrombin concentrations are key contributors to the balance of polymerization/degradation rates, they must be carefully considered when designing fibrin-based biomaterials, particularly for wound healing applications.15,25


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Commercial fibrin sealants, such as ‘Tisseel’ Baxter Healthcare Corporation and ‘Evicel’, Ethicon, work exceptionally well to stop blood loss in seconds.26–29 These sealants use extremely high levels of fibrinogen and thrombin to achieve fast clot polymerization dynamics; however, these high concentrations result in denser clots with reduced porosity.30 Consequently, cell infiltration into the wound bed does not begin until the sealant is degraded, thereby extending the length of time it takes for the ensuing stages of healing (including proliferation and inflammation) to occur compared to that observed in the presence of fibrin gels comprised of lower fibrinogen and thrombin concentrations.29 Because fibrin sealants are used in a multitude of surgical settings, including vascular, ophthalmic, orthodontic, colonic, gastric, breast, neuro-, orthopedic, and hepatic applications, enhancing the pro-healing capabilities of fibrin-based materials has the ability to improve patient outcomes in a myriad of clinical settings.29,31–34 Additionally, there are many conditions that result in unstable fibrin generation, such as hemophilia and oral anticoagulant-induced coagulopathies, which collectively affect ~5 million people in the US alone and cause delayed clot generation and wound healing.8,9,35–40 Improved fibrin-based materials could greatly improve healing outcomes for these patients as well. Additionally, the currently used sealants must be stored refrigerated and have a limited shelf-life, which minimizes their utility in emergency medicine and battlefield situations. In order to create a fibrin-based biomaterial that could be used to seal tissues quickly while maintaining a level of clot porosity that supports cell infiltration for the subsequent stages of wound healing, we propose the delivery of pre-polymerized fibrin nanoparticles, comprised of physiologically relevant fibrin concentrations, directly to wound sites. These stable fibrin nanoparticles can be lyophilized to simplify storage and transportation and rehydrated into a flowable format to facilitate clinical utilization. Additionally, the pre-formed fibrin nanoparticles



integrate with host fibrinogen to support physiological clot assembly at the wound site, with clot porosity and density compatible with appropriately timed biodegradation. We hypothesize, that due to their size, these particles can be diluted in saline and delivered to a number of wound sites, including dermal or internal wound/surgical sites, and once at the wound site, the particles can interact and support the native clotting cascade to generate fibrin scaffold in situ to support cell migration. Furthermore, because these particles are comprised of physiologically relevant densities of fibrin ligands, we expect that the nanoparticles will facilitate cell migration and enhance healing to a greater extent than bulk fibrin sealants that use supraphysiological levels of thrombin and fibrinogen.30 We also propose the use of fibrin nanoparticles as a carrier for keratinocyte growth factor (KGF) to enhance healing rates. The inflammation, proliferation and wound remodeling phases host an assortment of growth factors; important factors include isoforms of platelet derived growth factor (PDGF), transforming growth factor (TGF-β), epidermal growth factor (EGF), fibroblast growth factor (FGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). Granular tissue formation, cellular epithelialization and scar remodeling are influenced concurrently by growth factor and cellular functions at wound zones.13,41,42 Many growth factors have been utilized therapeutically for wound healing purposes; currently PDGF BB is FDA approved for human clinical use,43 and topical application of EGF, bFGF and interleukin-1 (IL-1) have been studied in humans for therapeutic use. We selected keratinocyte growth factor (KGF), also known as Fibroblast Growth Factor 7, for initial proof-of-principle experiments due to its demonstrated role in tissue repair, and cell proliferation; furthermore, KGF is approved by the FDA and induces genes that detoxify reactive oxygen species.18,19,44–48


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This report demonstrates the microfluidic in vitro synthesis of fibrin nanoparticles coupled with growth factor and the use of these nanoparticles to expedite dermal wound healing in a murine model. The controlled synthesis of fibrin particles in a microfluidic platform applies uniform shear in creating fibrin particulates and attempts to overcome heterogeneous fibrin polymerization typical of the conventional fibrin gelling process.26,49 In summary, fibrin nanoparticles coupled with KGF enhance tissue re-epithelialization and wound area closure.

Experimental Methods Cell lines, reagents and peptides Fibroblasts were used to characterize the effects of FBN and FBN-KGF on cell migration in vitro. Human dermal fibroblasts were acquired from ATCC. The cells were grown and maintained in high glucose Dulbecco’s Modified Eagle Media (DMEM) supplemented with sodium pyruvate (DML19 Caisson Labs, Smithfield UT), 10% v/v fetal bovine serum (ATCC, Manassas, VA), 10 mM L-glutamine (Cat# 091680149, MP Biomedicals, Santa Ana, CA) and penicillin-streptomycin (1% v/v) at 37°C and 5% CO2. Keratinocyte growth factor (KGF, CYT219, Prospec Protein Specialist, East Brunswick NJ) was covalently coupled to proteins using 1ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, (CAS 2595253-8 and CAS 106627-54-7, ThermoFisher Scientific, Waltham MA) for 2 hrs and quenched using Tris buffer under standard conditions. Synthesis of fibrin nanoparticles Human fibrinogen (1 mg/mL, depleted of plasminogen, von Willebrand factor and fibronectin FIB3, Enzyme Research Laboratories, South Bend, IN) was reacted with 0.5 U/mL human αthrombin (HT 1002a, Enzyme Research Laboratories, South Bend, IN) in HEPES buffer (25 mM HEPES, 150 mM NaCl, 5 mM CaCl2) pH 7.4 to generate fibrin. The reaction product (1 mg/mL



fibrinogen mixed with 0.5 U/mL thrombin in 2 mL volume of HEPES buffer) was prepared in a syringe and sheared into fibrin nanoparticles (FBN) by injecting it as the dispersive phase into a stream of synthetic silicone oil as the continuous phase (Acros Organics, AC63148-62-9, density 0.96 g/mL, viscosity 500 mPas at 25 °C) within a two-phase microfluidic droplet generator. The ratio of flow velocities of continuous and dispersive phases was controlled using two singlechannel Cole Palmer pumps (catalogue number 78-0100C). The microfluidic channels were made from laser-cut 3M tape (100 μm dispersive channel, 250 μm continuous channel, both with a height of ~400 μm) sandwiched between acrylic and glass sheets. The particles in the droplets were harvested by centrifugation at 10,000 G for 2 hours to remove silicone oil. Remaining oil was removed by subsequent resuspension of the nanoparticles and centrifugation at 15000 G for 30 minutes in 1.5 mL centrifuge tubes. The final suspension was dialyzed thrice in a 10 kDa slide-a-lyzer dialysis cassettes after dilution with more than 250 times the reaction volume each time Biophysical characterization of FBN Size and 3D structure of the FBN were characterized using dynamic light scattering (DLS), atomic force microscopy (AFM) and cryo-scanning electron microscopy (Cryo-SEM). The particle volume and surface charge of FBN were measured in a Zetasizer (Malvern, UK) and the 3D structure was visualized after freezing the particles in HEPES buffer and following CryoSEM (JEOL JSM 7600 FE SEM) sample preparation protocols by freezing the sample (FBN in 25 mM HEPES, 150 mM NaCl, 5 mM CaCl2 pH 7.4 buffer) in liquid nitrogen slush and transferring it under vacuum to the Alto-2500 chamber, where the sample is fractured at -95 C under 4 x 10-6 mbar vacuum. After cooling to -120 C, samples were coated with a layer of Au/Pd of ~5 nm thickness. Images were taken at 5 kV at 100,000 X magnification. Particle sizes


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were also determined under dry conditions using AFM. For AFM analysis, glass cover slips were dipped and cleaned in a series of solutions from alconox, distilled water, acetone, absolute ethanol and isopropanol. The cleaned glass cover slips were pretreated with 3aminopropyltrimethoxysilane (Sigma Aldrich, Missouri USA) washed and incubated with FBN 1 mg/mL while shaking at room temperature for 15 min. The cover slips were spun in a centrifuge and air-dried in a fume hood. The air-dried FBN were characterized using AFM (Asylum Research, Santa Barbara, CA), using AFM probes obtained from NanoAndMore USA (Watsonville, CA) and operated in air topography mode. The diameter and height trace of each air-dried particle (n > 50) were captured in Igor Pro and analyzed in ImageJ. Quantification of KGF and coupling to FBN To measure the amount of KGF coupled to FBN, lyophilized KGF (10 g) was suspended in 50 L reaction volume of HEPES (100 mM), TCEP (50 mM) buffer for 15 minutes, mixed with 1.5 nmol of Alexa Fluor 488 C5 maleimide (Invitrogen A10254), and shaken at room temperature for 4 hours. The reaction was quenched with β-mercaptoethanol (72 mM) following supplier protocols. The mixture was dialyzed in 10 KDa slide-a-lyzer cassettes to remove unbound fluorophore. Sample absorbance at 280 and 490 nm was measured using Thermo Scientific’s Nanodrop 2000 spectrophotometer. The labeling ratio was calculated following the Thermo Fisher and Invitrogen protocols as detailed in the Supplemental Information. FBN (400 g) were mixed with 10 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1 mg N-hydroxysulfosuccinimide, (CAS 25952-53-8 and CAS 106627-54-7, ThermoFisher Scientific, Waltham MA) in 50 mM 4-morpholinoethanesulfonic acid containing 100mM NaCl, pH 5.5(MES buffer) with a reaction volume of 400 L. After 30 min, the



suspension was centrifuged through a 30 KDa filter. The FBN were then mixed with KGF (4.5 g of unlabeled KGF mixed with 0.5 g Alexa 488 labeled KGF) in HEPES buffer (pH 7.4) for 3 hours. After quenching the coupling reaction with Tris buffer (100mM), the unreacted KGF 488 was removed by centrifuging with the 30 KDa filter. The FBN KGF 488 sample absorbance was measured at 280 and 490. The labeling ratio of KGF to FBN (487 nmol KGF per g FBN) was calculated from the labeling ratio of Alexa Fluor 488 to KGF (2.11 nmol label per nmol KGF) and 10% labeled Alexa Fluor 488 KGF to FBN (103 nmol label per g FBN). In the final batch of 400 L, the amount of FBN was 400 g and KGF was 195 pmol. Confocal microscopy The interaction between FBN and fibrin fibers in an in vitro clot was visually assessed using a confocal microscope. FBN (1 mg/mL) were incorporated with Alexa Fluor 488 fibrinogen during synthesis and then mixed with 10 mg/mL of fibrinogen and 1 mg/mL Alexa Fluor 594 fibrinogen and thrombin (0.1 and 0.5 U/mL) in a clot volume of 50 l. The clot was formed between a clean glass slide and a cover glass that was barricaded on either side by dual side transparent tape forming an enclosure for the clot. The edges of the cover glass were sealed with regular nail polish to prevent drying of the clot. The clot was imaged in a Zeiss LSM 880 Airyscan confocal microscope, C-Apochromat 40x/1.2 W objective lens, 0.44 m pinhole and excitation under 488 nm and 561 nm lasers. The acquisition channel filters included 488 ChS1 490-561 and ChS2 570-677. The images were acquired and analyzed in Zen software. Fibrin clot polymerization An in vitro fibrin polymerization assay was performed with 100 L fibrin reaction volumes.50 Fibrin gels were prepared by mixing fibrinogen (1 mg/mL) with thrombin (0.1 U/mL) in the presence or absence of lyophilized FBN (2 mg) in a 96 well plate and absorbance (OD) at


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350 nm was read over 200 minutes. The inverse of clot formation (1/OD at 350 nm) was plotted against inverse time (1/t min-1), and the data fit to a straight line with data points >15 of an average of 3 runs. The inverse of the slope (time/OD) gives rate of fibrin polymer formation. This inverse slope is compared between clot samples ± FBN. An average of 3 samples of each condition were analyzed. Traction force microscopy The interaction (adhesive traction) of human dermal fibroblasts with FBN was quantified using Traction Force Microscopy (TFM). FBN (0.2 mg/mL), bulk fibrin (0.2 mg/mL of fibrinogen treated with 0.5 U/mL thrombin at gel surface) or collagen (0.2 mg/mL) were immobilized overnight on the apical surface of sulfo-SANPAH (Proteochem, Hurricane, UT) activated 8.8 kPa polyacrylamide hydrogels (PA gels) at 4 C.51 Fluorescent microspheres (300 nm average diameter, Fluoro-Max, red fluorescent polymer microspheres, Thermo Fisher, Waltham MA) were embedded in the hydrogel, which served as fiduciary markers for traction measurements. After protein immobilization, the substrates were rinsed twice with 1X phosphate buffered saline (PBS), blocked with 1% w/v BSA in PBS for 10 min, rinsed, and sterilized by irradiation (365 nm) for at least 15 min before seeding cells. The fibroblasts were gently detached from the 75 cm2 flask using 3.5 mM ethylenediaminetetraacetic acid (EDTA) in PBS containing 1% w/v BSA.52–54 The cells were seeded at 10,000 cells/mL onto 8 mm diameter hydrogels, and allowed to adhere and spread for a maximum of 5 hr at 37°C under 5 % CO2. Under 20X magnification, 1.5 X NA, (EVOS fluorescent microscope), the fluorescent image of the bead positions were marked before and after cell detachment (3% SDS in PBS treatment). The absolute basal root mean square (RMS) traction force (BTF) was determined from fiduciary bead displacements, relative to the traction-free bead positions after cell removal using a custom MATLAB code.55–57



Constrained traction maps (from manually drawn cell boundary in MATLAB) and the RMS traction stress were determined from the bead displacement maps and compared. The means and standard error of the means were statistically computed for at least 5 samples from at least 3 separate experiments for each condition and significance compared using 2-way Anova (Prism). In vitro cell migration As previously described, a 3D wound healing assay was modified to determine the effect of FBN on human dermal fibroblast migration in vitro.58 Human dermal fibroblasts (~500,000 cells/mL) were embedded in 3D collagen gels (3 mg/mL). Using biopsy punches, “wounds” were created 2 mm in diameter and approximately 0.5 cm deep. The biopsied 3D wounds were separately filled with FBN, FBN-KGF, free KGF, HEPES or saline controls. The number of cells migrating into the hole from the surrounding 3D collagen gel was counted every 20-24 hours. The collagen gels were incubated with serum free DMEM media and data from each sample was normalized to its biopsy area (in mm2). Cell migration rate was measured by counting cell numbers within the normalized biopsy area (#cells per unit area) on each day. The values were plotted in Microsoft Excel, and each treatment (n > 5) was statistically compared with the saline control using the 2-way Anova (Prism). In vivo analysis of wound healing In vivo validation of the ability of FBN to assist wound healing was investigated in 10-weekold healthy male C57BL/6 mice (Charles River Laboratories Wilmington, MA) in a wellestablished full-thickness dermal injury model.59 Two epithelial dorsal dermal wounds were created on each mouse using a 4 mm diameter biopsy punch (day 0). The skin was excised and the inner subcutaneous panniculus carnosus was removed using surgical tools. Stitching the adjoining non-wounded skin to a sterilized silicone ring prevented outer skin contraction; this process promotes the wound to heal by re-epithelialization rather than contraction, which more


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closely mimics the process of human healing. A 4.9 mm inner diameter silicone ring was laser cut from a silicone sheet (acquired from Grace Bio-Labs, Bend, OR). The silicone ring was held in position with stationary glue and stitches. Care was taken that the glue did not touch the wound, but was in contact with remote edges of the silicone ring and outer skin. Each wound was immediately imaged using a handheld Nikon camera and separately treated with constant volume of 20 L each of treatment groups. The first study utilized treatment groups of control saline (0.9 % NaCl), bulk fibrin gel (20 mg/mL of fibrinogen, 0.5 U/mL thrombin in saline buffer), stock FBN solution (diluted in saline buffer), stock FBN-KGF with a loading capacity of 487 pmol KGF per mg FBN, and 1:10 and 1:100 dilutions of the stock FBNKGF solution in saline buffer. Our second study utilized treatment groups of control saline (0.9 % NaCl), bulk fibrin gel (20 mg/mL of fibrinogen, 0.5 U/mL thrombin in saline buffer), 1 mg/mL FBN-KGF with a loading capacity of 487 pmol KGF per mg FBN, 10 pmole of free KGF, and bulk fibrin mixed with 10 pmole of KGF. During the administration of each treatment, the absorption and drying of the agent on the wound area were not strictly controlled, but these times were quite short (< 5-10 minutes). After treatment, the wound was covered with translucent moisture responsive dressing (IV3000, Smith & Nephew, Andover, MA) attached over the silicone ring and not in direct contact with the wound. The dressing was laser cut to the required dimensions prior to surgery to ensure it properly matched the wound area. The wound area was imaged every day for 10 days using a handheld Nikon camera after removing the translucent dressing. A new translucent dressing was applied on the wound site after imaging each day. Image analysis of the wound was performed in ImageJ, and the scale was measured using a ruler (placed while taking the wound picture) (Figure S8). The % change in wound area on each day was normalized to its respective wound area in day 0 (day of biopsy). The rate of



wound healing was quantified by plotting averaged and % normalized wound area for each wound treatment. The slope of a linear fit of the average % normalized wound area for the first 3 days post biopsy was calculated in Prism and 2-way Anova statistics were performed to compare the slopes of each treatment. Tissue histology The mice were sacrificed on day 10 following approved protocols. The silicone ring and stitches were removed, and the final-day wound image was acquired using the Nikon camera with a manual ruler placed close to the wound. The wound area was excised, placed in a histology holding cassette and immediately immersed in 10% formalin solution for a minimum of 24 hours. The samples were immobilized into paraffin blocks, the blocks were sliced into 5-8 m thick sections, and the slices were transferred onto clean glass slides, mounted and dried. The samples were then deparaffinized, hydrated and stained following Martius yellow, brilliant crystal scarlet, aniline blue (MSB) staining procedure.60–62 The samples were washed thrice with xylene substitute – SafeClear II solution (Fisher Scientific, Hampton, NH; ten minutes each wash), followed with two 100% ethanol washes (three minutes each), one 95% ethanol wash (one minute) and one 80% ethanol wash (one minute). The deparaffinized samples were treated with Bouin’s fixative solution (Ricca Chemical, Arlingotn TX, Catalogue number 1120-16) and heated to 60 C for an hour (note: wear face mask in this step) and then washed in running tap water (five minutes) until all color disappeared from tissue. The samples were treated with Weigert hematoxylin (0.5% hematoxylin, 14.5% iron chloride in 47.5% ethanol) for seven minutes and washed in running tap water for one minute. The samples were rinsed twice in 95% ethanol for ten seconds and stained with Martius yellow solution (0.25% Martius yellow and 2% phosphotungstic acid in 95% ethanol) for two minutes. The samples were rinsed again with 95%


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ethanol for ten seconds twice and stained with Chromotrope 2R solution (1% Chromotrope 2R in 2.5% acetic acid) for ten minutes. The samples were washed twice in distilled water for 10 seconds, placed in 1% phosphotungstic acid with gentle shaking of slides and washed again in distilled water. The samples were then stained in 0.5% aniline blue solution (prepared in 1% acetic acid solution) and incubated for two minutes. After rinsing in distilled water, the samples were dehydrated by washing with varying grades of alcohol (70%, 80%, 95% and absolute alcohol) followed by Safeclear II treatment. The samples were then covered with a coverslip using mounting media, air-dried overnight and imaged in EVOS microscope. The acquired images were analyzed in ImageJ. Results Fabrication of FBN Fibrinogen (1 mg/mL) polymerized with thrombin (0.5 U/mL) in HEPES buffer was used to generate a fibrin hydrogel. This fibrin hydrogel (dispersive phase) was sheared in a Y-shaped fibrin droplet generator with a flow of silicone oil (continuous phase).63–65 We controlled the relative flow velocities in the two phases to control the droplet size. In practice, we used flow rates at the two pumps of 1 mL/hr (continuous phase) and 0.1 mL/hr (dispersive phase) to produce flow velocities within the channels of ~0.16 m/min for the silicone oil and ~0.04 m/min for the fibrin hydrogel. The fibrin droplets pinched off at the Y intersection and were collected under stirring at room temperature. The material used to form the microfluidic droplet channel was laser-cut, dual-sided 3M tape 9495B (Figure 1A), which was sandwiched between a glass slide and an acrylic sheet. After shearing into droplets, the fibrin particulates (FBN) produced a cloudy suspension at room temperature. After removing the oil by washing FBN, the final suspension appeared clear. The purified FBN were then resuspended in HEPES buffer pH 7.4 in 2 mL and stored at -20C. No aggregates were observed under these conditions. For confocal



experiments the synthesized FBN were dialyzed in water, aliquoted and stored in -20 °C after lyophilization. The average volumetric diameter of hydrated FBN was measured to be 600 ± 200 nm in DLS. Air-dried FBN were characterized using AFM (air topography mode); the dehydrated FBN exhibited a spread and flat structure with a diameter of 370 ± 235 nm and a height of 15 ± 14 nm (n = 60, representative Figure 1C).


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Figure 1. FBN characterization. A-B) The design and working model of the Y-shaped microfluidic droplet generator used to generate fibrin nanoparticles (FBN) is shown. C) The size of harvested FBN is characterized as a dry particle in Atomic Force Microscopy (AFM). D) typical CryoSEM images of hydrated FBN are shown.

In vitro biological activity of FBN Since FBN are synthesized from fibrinogen and thrombin, the ability of FBN to integrate with a fibrin network during clot formation was investigated by mixing 0.2 mg lyophilized labeled FBN with 10 mg/mL of labeled fibrinogen in the presence of 0.1 U/mL thrombin. Confocal images of Alexa Fluor 488 labeled FBN, mixed with Alexa Fluor 594 labeled fibrinogen in the presence of thrombin, showed a thick layer of fibrin fibers attached to the FBN surface. Lyophilized FBN (labeled with Alexa Fluor 488 fibrinogen during synthesis) aggregated to a bigger mass, and z-stack images in confocal microscopy suggest envelopment of FBN (green) by fibrinogen (red fibers) in the presence of thrombin (Figure 2 A-B). This data suggests that FBNs have active fibrin knobs that support fibrin aggregation, a mechanism that supports in vivo physiological clot formation and integration with the wound. Fibrin clots formed in the presence and absence of FBN are presented in Figure S2 A, C. As a control, lyophilized FBN (200 g) incubated overnight with fibrinogen (10 mg/mL) but in the absence of thrombin, neither formed a clot nor was coated with fibrin (Figure S2 D). How FBNs influence the dynamics of fibrin polymerization was also investigated through the use of an absorbance based fibrin polymerization assay. In this assay, 2 mg of lyophilized FBN was mixed with 1 mg/mL fibrinogen and 0.1 U/mL thrombin in a reaction volume of 100 μl and absorbance at 350 nm was measured over 200 minutes. The A350 values overtime for FBN+


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fibrinogen + thrombin, fibrinogen + thrombin, and fibrinogen - thrombin, are shown in Figure S1 A. We additionally produced a plot of 1/absorbance vs 1/ time and performed linear regression, shown in Figure S1 B. The inverse of the slope obtained from this plot is the rate at which the opacity of the clot is increased, which is the rate of polymerization. The inverse slope was 0.109 (OD/time) with FBN compared to 0.039 (OD/time) in their absence (Figure S1 A-B), indicating that in the presence of FBNs, the final polymerization state was reached faster than in the absence of FBN. We also found that the maximum final turbidity of the polymerized fibrin was higher in the absence of FBNs.



Figure 2. FBN integrates with fibrin clot. A) FBN labeled with Alexa Fluor 488 fibrinogen (green FBN) were lyophilized and then mixed with Alexa Fluor 594 labeled fibrinogen in the presence of low thrombin (0.1 U/mL). A 2D image of red fibrin fibers forming around FBN is shown. B) A 3D stack of fibrin fibers around FBN particle is shown for greater detail. A zoomed-in image of fibrin fibers is shown in C-D. No fibers were observed in FBN incubated with fibrinogen in the absence of thrombin (Figure S2).


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Since fibrin has active ligands that interact and bind with integrin receptors,16,17,18,19 we investigated whether fabrication of fibrin into FBN can still support human dermal fibroblast (HDF) adhesion and measurable traction generation. We tested the cell adhesive function of FBN by chemically coupling FBN to the surface of an antifouling non-cell adherent polyacrylamide (PA) hydrogel. HDF attachment and spreading was measured after exposure to FBNfunctionalized polyacrylamide gels (FBN-PA gels). The extent of cell spreading and traction force generation was measured within 5 hours of cell exposure to FBN-PA gels. The positive controls included polyacrylamide gels functionalized with collagen or bulk fibrin. Human dermal fibroblasts attached onto FBN-PA gels, and the extent of spreading was 2200 ± 1600 μm2 on FBN surfaces, which was not significantly different from collagen (3400 ± 1100 μm2) or bulk fibrin (2400 ± 1000 μm2, Figure 3 A). Control cell images included HDF grown on naked and 1% bovine serum albumin functionalized PA gels. No cells bound to naked PA gels and very few round cells (not spread and weakly attached) were observed in BSA coated PA gels (Figure S3). The traction force a single cell exerts on its substrate is an indirect indicator of its intracellular contractility.52,66,67 Measuring the basal traction stresses of single cells on a functionalized polyacrylamide substrate provided an indirect comparison of the effect of the FBN on dermal fibroblast contractility and the ability to exert forces on the substrate. Human dermal fibroblast’s contractility on different functionalized substrates (FBN, collagen and bulk fibrin) was compared using traction force microscopy. Cells on FBN-coated surfaces produced a traction of 233 ± 150 Pa (n=11), while on collagen and bulk fibrin gels, the cells produced 260 ± 133 Pa (n=23) and 165 ± 105 Pa (n=5), respectively (Figure 3 B-C). The data were not significantly different from one other, suggesting that dermal fibroblasts were adhering onto FBN equivalently well when compared to collagen or bulk fibrin gels.



5000

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-20

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Figure 3. FBN interact with human dermal fibroblasts. A) Human dermal fibroblasts (HDF) adhesion and extent of spread area on polyacrylamide gels functionalized with collagen or FBN


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or bulk fibrin clot is compared. To minimize the host cell modifying the matrix environment the cells were exposed to gels for five hours. B) The extent of HDF traction is compared on different substrates, including polyacrylamide gels functionalized with collagen, FBN or bulk fibrin. C) Representative heat maps of traction vectors under collagen and FBN gels are shown. FBN coupled with KGF enhance cell migration and wound healing Keratinocyte growth factor (KGF) is a well-known growth factor involved in tissue healing. KGF is of mesenchymal origin and enhances the regenerative capacity of epithelial tissues.8,18,19,44 We hypothesized that administering FBN carrying KGF to wound sites would enhance the rate of the cellular re-epithelialization and wound healing compared to FBNs alone. KGF was covalently attached to FBN and administered to in vitro 3D wounds by adding 20 l of sample into the biopsied region of the collagen matrix (Figure 4) and to in vivo dermal wounds in mice (Figure 5). In both models, the FBN-KGF enhanced cell migration and wound closure. In the collagen wound model, FBN-KGF increased the total number of cells migrating into the wound zone. Cell number was measured every day and normalized to wound area. Comparing treatment conditions of FBN, FBN-KGF, or free KGF with saline, FBN-KGF had significantly higher cell numbers in the collagen wound (Figure 4). Experiments comparing the influence of varying FBN dosages are shown in Figure S4. In these studies, saline and HEPES control treatments showed an average of 5 ± 2 and 4 ± 2 cells migrated into the wound area (per mm2 area) after 3 days, respectively. Similarly, for FBN introduced into the in vitro “wound”, 1:1, 1:10, and 1:100 dilutions of the stock FBN solution gave averages of 6 ± 2, 7 ± 3, 5 ± 3 cells/mm2 area and 1:1, 1:10, and 1:100 dilutions of FBN-KGF gave averages of 22 ± 7, 8 ± 2, and 7 ± 2 cells/mm2 area, respectively (Figure S4). FBN-KGF, showed significantly enhanced cell recruitment in 3D collagen gels compared with saline and HEPES controls. Both



saline and HEPES buffers were used as controls to evaluate if differences in migration were observed between the two buffers utilized for in vitro and in vivo experiments. Overall, these results showed that FBN alone is not sufficient to enhance cell migration. It appears that a chemotactic agent is required in order to induce cell migration into the wound bed. However, we do show that FBNs do not inhibit cell migration (Figures 4 and S4), as bulk fibrin sealants have previously been reported to do.26–29 Bulk fibrin sealants require high concentrations of fibrinogen and thrombin in order to induce fast polymerization at the surgical/wound site; by delivering prepolymerized FBNs we are able to deliver the fibrin much more easily and without inhibiting migration.


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Figure 4. FBN-KGF support cell migration. A) A cartoon of 3D collagen wound experimental model is presented. B) The number of cells migrating into the created 3D collagen wound area is statistically compared by administering the wound zone with FBN (20 g / wound), FBN KGF (20 g FBN loaded with 10 pmol KGF) and free KGF (3.5 pmol). Saline is used as control.



For in vivo dermal wounds, the tissue re-epithelialization and wound-healing phenomenon was indirectly measured as a % decrease in wound area post tissue biopsy.68,69 The rate and slope of healing (assuming an initial linear healing) for the first 3 days after wound creation is compared for each treatment after normalization to the initial wound area (Figure 5). FBN-KGF (20 g FBN carrying 10 pmol KGF, n = 7), closed dermal wounds with a 3 day average dermal area closure of 48 ± 9 %, compared to Saline (n = 7, 35 ± 20 %), bulk fibrin (400 g fibrin, n = 6, 34 ± 9 %), bulk fibrin mixed with 10 pmol KGF (400 g fibrin + 10 pmol KGF, n = 5, 32 ± 16%), and free KGF (10 pmol, n = 9, 42 ± 12%). Assuming a linear healing rate of the first 3 days, the slopes of FBN-KGF was -16 ± 3, compared with saline (-12 ± 0.3), bulk fibrin (-11 ± 3), bulk fibrin mixed with KGF (-11 ± 2) and free KGF (-14 ± 2). We attempted to conjugate KGF to bulk fibrin to directly compare how covalent coupling of KGF to bulk fibrin compared to covalent coupling of KGF to FBNs. Unfortunately, we found that the coupling protocol consistently resulted in aggregation of the product, therefore we were not able to directly compare KGF covalently coupled to bulk fibrin gels with KGF covalently coupled to FBNs. Influence of dilutions of FBN KGF on dermal closure was separately compared (another set of mice dermal injury models, with similar treatment volumes of 20 L) and reported in Figure S5, where the control saline treatment (n=5) had an average wound coverage of 18 ± 10%, while FBN (1:1, n=4) had 15 ± 16% and FBN KGF (1:1 (n=6), 1:10 (n=5), 1:100 (n=5), loaded with ~487 pmol KGF / mg FBN) healed with an average coverage of 46 ± 12%, 33 ± 13% and 29 ± 9% respectively. The % wound area healed using bulk fibrin gels (20 mg/mL of fibrinogen with 0.5 U/mL thrombin) was 27 ± 33%. The negative slope (assuming a linear healing rate) of average normalized three-day healing under treatments with saline, plain FBN, or bulk fibrin was - 5.9, - 6.4, and - 7.8, respectively. In contrast, FBN KGF at 1:1, 1:10, 1:100 expedited healing


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rates in the first three days of treatment to -11.5, -11. 4 and -12.4, respectively. The slopes produced by the FBN KGF treatment were statistically different (*p < 0.05) from saline treatment, FBN and bulk fibrin treatment (Figure S5 B-C) using 2-way Anova.

A Bulk FNG

Bulk FNG FBN-KGF +KGF

KGF

Day 0

Saline

Day 3

B % Normalized wound closure

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


KGF Bulk FNG+KGF FBN-KGF

Sal Bulk FNG

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100

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0

1

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Figure 5. FBN-KGF support dermal wound healing. A) Effect of fibrin nanoparticles coupled with KGF on wound re-epithelization is compared with saline, bulk fibrin, free KGF, and KGF mixed with bulk fibrin. Representative mice dermal wound images are shown. (B) The effect of coupling KGF, FBN-KGF (20 g FBN carrying 10 pmol KGF in a volume of 20 L) is compared with, bulk fibrin gels (Bulk FNG - 400 g fibrinogen treated with 0.5 U/mL thrombin and applied immediately over the wound), Bulk fibrin + KGF (400 g fibrinogen mixed with 10



pmol KGF and 0.5 U/mL thrombin) and saline treatment. The reduction (healing) of wound area is normalized to day 0 of the wound in mm2. Assuming a linear healing rate, the slope of wound area reduction over 3 days are compared.

After 10 days, histological analyses of treated wounds showed thicker epidermal layers when wounds were treated with KGF and FBN-KGF compared with saline treatment. The treatment with FBN-KGF and KGF show enhanced cellularity at epidermal layers (Figure 6). Granulation and cell infiltration is a good indicator of wound healing. In sections obtained following treatment with bulk fibrin, we frequently observed thick fibrin layers remaining on top of the wound site, indicative of lack of degradation of the fibrin gel and in many cases the epithelial layer appeared to be forming on top of the gel, indicating that cells were migrating on top of the gel and not infiltrating. A zoomed-out image of a bulk fibrin sample is shown in Figure S7 to show a larger region of the remaining fibrin clot, shown in red. This region appears fragile as it was easily disrupted during sectioning. Histological evaluation of tissues collected from our initial dose response studies are shown in Figure S6. In the 1:10 FBN-KGF samples, we frequently observed very thin epidermal regions, that were quite fragile, that were easily removed during sectioning.


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Figure 6. KGF support epidermal growth and cellularity at wound zones. Histology of healthy unwounded skin is compared to wounded skin ten days after a single treatment with saline, bulk fibrin, free KGF, FBN-KGF, or bulk fibrin mixed with KGF. Representative images of tissue samples stained with MSB are shown. The epidermal layer is marked with dashed white line. The black line indicates a scale of 400 m. Discussion There is a growing demand to treat wounds efficiently as the number of patients suffering from chronic non-healing wounds is increasing at an alarming rate. Fibrin is well-investigated and FDA approved as a tissue sealant and to stop external bleeding, but is used at a very high density that may retard wound healing.1,26 To address issues of delayed rates of wound healing, fibrin nanoparticles were generated from bulk fibrin, prepolymerized at low density, using a two-phase microfluidic droplet generator. The method utilized here-in could be easily scalable, for example



by introducing multiple dispersive channels into the continuous channel or by implementing multiple droplet generators operating in parallel, in order to generate sufficient materials for clinical translation.70 We hypothesized, that due to their size, these particles can be diluted in saline and delivered to a number of wound sites, including dermal or internal wound/surgical sites, and once at the wound site, the particles can interact and support the native clotting cascade to generate fibrin scaffold in situ to support cell migration. In vitro analyses suggest that FBN can integrate into a clot and support fibroblast adhesion and traction generation. We expect that even in non-actively bleeding wounds, such as those experienced in the extremities of diabetics, the FBN will interact with the small amount of nascent fibrin fibers available in the wound bed to generate a provisional matrix scaffold capable of supporting cell migration, and such applications will be explored in future studies. FBN can act as a carrier for biomolecules; when coupled with keratinocyte growth factor, FBN enhanced fibroblast cell migration in an in vitro 3D wound model and supported quicker wound healing in a murine dermal model. The biochemical mechanism by which the FBN supports cell migration and wound closure merits further investigation. Early results of FBN interacting with fibrinogen in the presence of low levels of thrombin suggests that active knobs in fibrin on the surface of the nanoparticles support ongoing fibrinogen assembly around the FBN and improve fibrin polymerization rate. FBN enhance the efficacy of fibrin polymerization rate (Figure S1) but results in a lower final clot turbidity (Figure S1). This phenomenon may be due to altered fibrin mesh architecture (confocal results, Figure 2) or fibrin polymerization dynamics (biochemical assay, Figure S1) that warrants further investigation of the biochemical interactions between FBN and native fibrin polymerization in future studies.


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FBN alone do not enhance cell migration or dermal wound closure and requires KGF to promote enhanced cell migration. Most importantly, the enhanced cell recruitment in vitro, increased wound area closure rate and increased epidermal layer thickness support the value of the FBN-KGF treatment for expediting early wound healing.41,71 KGF is a pro-migratory growth factor that promotes healing.18,19,44–47 KGF is strongly upregulated in wounded dermal tissue and is secreted by stromal fibroblasts. KGF binds to the KGF receptor (KGFR), which is a tyrosine kinase receptor, on keratinocytes. Due to its important role in promoting keratinocyte migration into the wound bed and its pro-healing action, KGF has been applied topically to promote healing.46,72,73 In the wound models used here, free KGF does enhance cell migration in vitro (Figure 4) and increase wound healing rates in vivo (Figure 5). Most importantly, FBN-KGF promotes cell migration to a greater degree than free KGF alone (Figure 4) and increases wound closure rates to a greater degree than KGF alone (Figure 5). This could be because attachment of KGF to FBNs presents the KGF to the cells more efficiently, delivers KGF over a more extended period, or stabilizes the KGF for a longer period of time at physiological temperatures. Considering the need for faster wound healing therapies and the disadvantages of bulk fibrin gels, the application of fibrin nanoparticles will be advantageous in clinical practice, as FBN could be mass produced in a microfluidic platform and can be coupled to wound healing peptides and growth factors. Compared to bulk fibrin gels, FBNs with physiologically relevant fibrinogen and thrombin concentrations, provide a simpler method for application to wounds, compared to the application of bulk fibrin sealants, and should produce more effective wound treatment strategies. Commercial fibrin sealants are typically delivered in a double barreled syringe format that mix fibrinogen and thrombin directly at the wound site and result in rapid polymerization. The rapid rate of polymerization means that the health professional applying the material must



move very fast to avoid polymerization in improper areas and/or clogging in the tube. This requires a great deal of skill. Because our particles are prepolymerized and flowable, they avoid this limitation of traditional fibrin sealants. Even though FBN are not expected to quickly stop major blood loss in a damaged tissue, due to the low fibrinogen and thrombin concentrations used, FBN can function to support wound healing post hemostasis. The issues with commercial bulk fibrin sealants, including storage (fibrinogen and thrombin temperature sensitivity), transportation (shelf life in transit), device development (double barreled syringe) and clinical difficulty in administering fibrinogen and thrombin at high concentrations, can potentially be overcome by providing premade fibrin nanoparticles, in the form of FBN, that carry woundhealing factors, such as KGF. Even though FBN is not expected to quickly stop blood loss in a damaged tissue, due to the low fibrinogen and thrombin concentrations used, FBN can function to support wound healing post hemostasis. The issues with commercial fibrin sealant’s storage (fibrinogen and thrombin temperature sensitivity), transportation (shelf life in transit), device development (double barreled syringe) and clinical difficulty in administering fibrinogen and thrombin at high concentrations as a bulk fibrin sealant can be potentially overcome by providing premade fibrin nanoparticles, in the form of FBN, that carry wound-healing factors, such as KGF. Conclusions In conclusion, we fabricated fibrin nanoparticles at room temperature using a simple two-phase microfluidic system that could be mass-produced. The fibrin nanoparticles coupled to keratinocyte growth factor supported dermal fibroblast chemotaxis to wound zones in vitro and enhanced the healing of mice dermal wounds in vivo. These fibrin nanoparticles may be useful as an alternative for fibrin sealants to promote healing.


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Author Contributions I.M. designed and conducted experiments, analyzed data, and drafted the manuscript. E.S. assisted in animal experiments. F.L. designed experiments, provided technical advice and edited the manuscript. A.C.B. designed experiments, analyzed data, edited, wrote and finalized the manuscript. We would like to thank Daniel Chester, Seema Nandi, Mario Castaneda, Francis Kim and Susan Bernacki for guidance in using the AFM, implementing the 3D in vitro wound healing model, cryoSEM and providing histology expertise respectively. This work was performed in part at the Analytical Instrumentation Facility (AIF) at NCSU, which is supported by the State of North Carolina and the National Science Foundation (ECCS-1542015). The AIF is a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), a site in the National Nanotechnology Coordinated Infrastructure (NNCI). The authors acknowledge Elaine Zhou at the AIF for assistance with CryoSEM, and Mariusz Zareba at the CMIF, for assistance with confocal microscopy. Funding was provided by the Ross Lampe Chair in Biomedical Engineering, NC State University start-up funds, and the American Heart Association 16SDG29870005. Supplemental Information The supplemental information contains the method for calculating the protein concentrations of KGF and FBN and Supplemental Figures describing additional data. References (1)

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Graphical Abstract


Fibrin nanoparticles coupled with keratinocyte growth factor enhance

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